This application is a 371 application of PCT/JP00/00572 filed Feb. 2, 2000.
This invention relates to a 2-N-substituted or unsubstituted-2-amino-5-methylpiperidine-3,4-diol which is a novel compound having an inhibitory activity against glycosidases, or pharmaceutically acceptable salts thereof. This invention also relates to a process for the preparation of such a novel compound. Furthermore, this invention relates to a novel glycosidase inhibitor consisting of such a novel compound. Moreover, this invention relates to a pharma-ceutical composition comprising as an active ingredient said 2-N-substituted or unsubstituted-2-amino-5-methylpiperidine-3,4-diol having an inhibitory activity against glycosidases. Besides, this invention includes an intermediate compound to be used for the synthesis of said novel compound.
Various glycosidases which are a glycoside hydrolase are an enzyme which is distributed widely in animal cells, microorganisms, viruses, and so on. In mammalian animals, it has been considered that glycosidases control a great variety of physiological mechanisms, including oncogenesis, metastasis of cancer cells, viral or bacterial infection, inflammation, immunological functions, fertilization of an ovum, and others, in which carbohydrate chains of glycoproteins and glycolipids can participate through the carbohydrate metabolism. Moreover, certain glycosidases participate in the digestive mechanism of food through the degradation of polysaccharides such as starch and sucrose, and oligosaccharides. Furthermore, it has been found that such a substance, which is inhibitory to glycosidases capable of liberating the carbohydrate chains bound to the surface of a cell membrane, is possible to have an immunomodulating action, an action of controlling inflammation and an action of controlling the metastasis of cancer cells, as well as an action of controlling infection of an AIDS virus or an influenza virus. Moreover, such substances that are inhibitory to glycosidases having catabolism which can participate in the digestive mechanism of food are found to be important, since they are useful as an antidiabetic agent or an agent for obesity.
Thus, in view that glycosidases are an enzyme which is important in the living body, it is also important to study physiological properties of glycosidases. In the study of the properties of glycosidases, use can be made of a substance having an action which inhibits the enzymatic activities of glycosidases. Moreover, it can be expected that certain glycosidase-inhibitory substances can be utilized as an inhibitor to the metastasis of cancer cells, and chronic articular rheumatism.
Therefore, it has been keenly demanded to provide such a novel compound which is of low toxicity, which is water-soluble and which has a potent inhibitory activity against glycosidases.
It is an object of this invention to provide a novel compound having a potent inhibitory activity against glycosidases, and also to provide a novel glycosidase inhibitor. Moreover, it is another object of this invention to provide a process for the preparation of such a novel compound.
In order to achieve the above-mentioned objects of this invention, we, the present inventors, have paid attention to 1-N-iminosugars which are active as a glycosidase inhibitor, and we eagerly have made research about these 1-N-iminosugars. As a result, the present inventors have now succeeded in synthesizing several novel piperidine derivatives which have a potent inhibitory activity against glycosidases and which are represented by the general formula (I) given hereinafter. Moreover, the present inventors have now found a new preparation process which is able to synthesize efficiently the novel piperidine derivatives of the general formula (I). Based on these findings, the present inventors have completed this invention.
According to the first aspect of this invention, therefore, there is provided (2R,3S,4R,5R)-2-amino-5-methypiperidine-3,4-diol or a (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol represented by the general formula (I): 
wherein R1 and R2 each are a hydrogen atom, or R1 is a hydrogen atom and R2 is a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group, or R1 and R2 together denote a phthaloyl group, or a pharmaceutically acceptable salt thereof.
The salt of the compound of the general formula (I), which is provided according to the first aspect of this invention, includes acid addition salts at the imino group of the compound of the general formula (I). Such acid addition salts include, in particular, such pharmaceutically acceptable acid addition salts of said compound with a pharmaceutically acceptable inorganic acid such as hydrochloric acid and sulfuric acid or a pharmaceutically acceptable organic acid such as acetic acid, propionic acid, and the like.
In the compound of the general formula (I), the lower alkanoyl group or lower xcfx89-trihaloalkanoyl group mentioned for the definition of R2 is preferably acetyl group, trifluoroacetyl group or trichloroacetyl group, but generally may be the known lower alkanoyl groups or lower xcfx89-trihaloalkanoyl groups which are conventionally used in chemistry of an amino sugar.
Examples of the compound of the general formula (I), which is provided according to the first aspect of this invention, include such compounds given in the following (1) to (5):
(1) (2S,3S,4R,5R)-2-acetamido-5-methylpiperidine-3,4-diol represented by the formula (Ia): 
(2) (2S,3S,4R,5R)-2-trifluoroacetamido-5-methyl-piperidine-3,4-diol represented by the formula (Ib): 
(3) (2S,3S,4R,5R)-2-trichloroacetamido-5-methyl-piperidine-3,4-diol represented by the formula (Ic): 
(4) (2S,3S,4R,5R)-2-phthalimido-5-methylpiperidine-3,4-diol represented by the formula (Id): 
and
(5) (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol represented by the formula (Ie): 
Next, there are described the physicochemical properties of (2S,3S,4R,5R)-2-acetamido-5-methylpiperidine-3,4-diol of the formula (Ia), (2S,3S,4R,5R)-2-trifluoro-acetamido-5-methylpiperidine-3,4-diol of the formula (Ib), (2S,3S,4R,5R)-2-trichloroacetamido-5-methylpiperidine-3,4-diol of the formula (Ic), (2S,3S,4R,5R)-2-phthalimido-5-methylpiperidine-3,4-diol of the formula (Id) and (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol of the formula (Ie), among the compound of the general formula (I) which are provided according to the first aspect of this invention.
1. Hydrochloride of (2S,3S,4R,5R)-2-Acetamido-5-methylpiperidine-3,4-diol [the Compound of the Formula (Ia)]
Color and Appearance: Colorless amorphous solid; Molecular formula: C8H16N2O3.HCl; Specific rotation: [xcex1]27Dxe2x88x9232.3xc2x0 (c 0.41, methanol); 1H-NMR spectrum (CD3OD, xcex4 ppm): 1.05 (3H, d, J=6.8 Hz, xe2x80x94CH3), 1.95-2.06 (1H, m, H-5), 2.05 (3H, s, xe2x80x94NHCOCH3), 2.92 (1H, dd, J=12.2 and 3.9 Hz, H-6eq), 3.05 (1H, br t, J=12.5 Hz, H-6ax), 3.72 (1H, dd, J=10.3 and 2.4 Hz, H-3), 3.84-3.87 (1H, m, H-4), 4.91 (1H, d, J=10.3 Hz, H-2); IR spectrum (KBr): 3325, 1660, 1460, 1400, 1140, 1060, 910 cmxe2x88x921; Mass spectrum (FAB-MS): m/z 189.2 (M+H)+, 176.1, 154.1, 137.1, 120.1, 107, 89, 77.
2. Hydrochloride of (2S,3S,4R,5R)-2-Trifluoroacetamido-5-methylpiperidine-3,4-diol [the Compound of the Formula (Ib)]
Color and Appearance: Colorless amorphous solid; Molecular formula: C8H13N2O3F3.HCl; Specific rotation: [xcex1]28Dxe2x88x9242.3xc2x0 (c 0.46, methanol); 1H-NMR spectrum (CD3OD, xcex4 ppm): 1.06 (3H, d, J=6.8 Hz, xe2x80x94CH3), 1.95-2.06 (1H, m, H-5), 2.97 (1H, dd, J=12.2 and 4.4 Hz, H-6eq), 3.09 (1H, br t, J=12 Hz, H-6ax), 3.84 (1H, dd, J=10.3 and 2.4 Hz, H-3), 3.87-3.90 (1H, m, H-4), 4.99 (1H, d, J=10.3 Hz, H-2) IR spectrum (KBr): 3440, 3250, 2950, 2780, 1730, 1555, 1400, 1260, 1220, 1180, 1100, 990, 900 cmxe2x88x921; Mass spectrum (FAB-MS): m/z 243.2 (M+H)+, 154.1, 137.1, 120.1, 107.1, 89, 77.
3. Hydrochloride of (2S,3S,4R,5R)-2-Trichloroacetamido-5-methylpiperidine-3,4-diol [the Compound of the Formula (Ic)]
Color and Appearance: Colorless amorphous solid; Molecular formula: C8H13N2O3Cl3.HCl; Specific rotation: [xcex1]23Dxe2x88x9237.8xc2x0 (c 0.23, methanol); 1H-NMR spectrum (CD3OD, xcex4 ppm): 1.07 (3H, d, J=6.8 Hz, xe2x80x94CH3), 1.96-2.02 (1H, m, H-5), 2.99 (1H, dd, J=12.2 and 4.4 Hz, H-6eq), 3.07 (1H, br t, J=12 Hz, H-6ax), 3.88-3.91 (1H, m, H-4), 3.94 (1H, dd, J=10.3 and 2.4 Hz, H-3), 4.97 (1H, d, J=10.3 Hz, H-2); Mass spectrum (FAB-MS): m/z 293 (M+H)+, 291.08 (M)+, 170.18, 154.09, 136.09, 130.13, 112.06, 107.04, 89.03, 77.04.
4. Hydrochloride of (2S,3S,4R,5R)-2-Phthalimido-5-methylpiperidine-3,4-diol [the Compound of the Formula (Id)]
Color and Appearance: Colorless amorphous solid; Molecular formula: C14H16N2O4.HCl; Specific rotation: [xcex1]23Dxe2x88x9219.6xc2x0 (c 0.25, methanol); 1H-NMR spectrum (CD3OD, xcex4 ppm): 1.11 (3H, d, J=6.8 Hz, xe2x80x94CH3), 2.14-2.23 (1H, m, H-5), 3.10 (1H, dd, J=12.2 and 4.4 Hz, H-6eq), 3.23 (1H, br t, J=12 Hz, H-6ax), 3.99 (1H, br s, H-4), 4.54 (1H, dd, J=10.3 and 2.4 Hz, H-3), 5.44 (1H, d, J=10.3 Hz, H-2), 7.88-8.00 (4H, m, phthalimido); Mass spectrum (FAB-MS): m/z 277.19(M+H)+, 265.17, 202.22, 170.17, 154.10, 136.09, 130.13, 107.04, 89.03, 77.04.
5. Hydrochloride of (2R,3S,4R,5R)-2-Amino-5-methyl-piperidine-3,4-diol [the Compound of the Formula (Ie)]
Color and Appearance: Colorless amorphous solid; Molecular formula: C6H14N2O2.HCl; Specific rotation: [xcex1]27Dxe2x88x9224.90 (c 0.15, methanol); 1H-NMR spectrum (CD3OD, xcex4 ppm): 1.04 (3H, d, J=6.8 Hz, xe2x80x94CH3), 1.95-2.02 (1H, m, H-5), 2.99-3.04 (2H, m, H-6), 3.55 (1H, dd, J=2.5 and 8.8 Hz, H-3), 3.84 (1H, br t, J=2.5 Hz, H-4), 4.42 (1H, d, J=8.8 Hz, H-2); 13C-NMR spectrum (CD3OD, xcex4 ppm): 14.21 (CH3), 33.52 (C-5), 44.26 (C-6), 72.25 (C-3 or C-4), 72.99 (C-4 or C-3), 88.86 (C-2); Mass spectrum (FAB-MS): m/z 130.2 (Mxe2x80x94NH3+H)+, 107.0, 89.0, 77.1.
The following Test Examples demonstrate that each of (2S,3S,4R,5R)-2-acetamido-5-methylpiperidine-3,4-diol of the formula (Ia), (2S,3S,4R,5R)-2-trifluoroacetamido-5-methylpiperidine-3,4-diol of the formula (Ib), (2S,3S,4R,5R)-2-trichloroacetamido-5-methylpiperidine-3,4-diol of the formula (Ic), (2S,3S,4R,5R)-2-phthalimido-5-methylpiperidine-3,4-diol of the formula (Id), and (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol of the formula (Ie) according to this invention has the glycosidase-inhibitory activity.
This Test Example is a test example for determining the xcex1-fucosidase-inhibitory activity of the compound of the general formula (I) according to this invention.
Assay of the xcex1-fucosidase-inhibitory activity was conducted according to a modification of the method described in the xe2x80x9cJournal of Biological Chemistryxe2x80x9d, 252, pp.5194-5200 (1977).
Thus, 0.5 mL of 0.025M citrate-phosphate buffer (pH 6.3), 0.1 mL of the same buffer containing 1.5 mM p-nitrophenyl xcex1-L-fucopyranoside dissolved therein, and 0.1 mL of either water or an aqueous solution containing as the tested compound any one of the compounds of the formulae (Ia), (Ib), (Ic) and (Id) according to this invention were mixed with each other and pre-incubated in a 96-well titer plate at 37xc2x0 C. for 10 minutes. After the completion of the pre-incubation, to the pre-incubated mixture was added 0.01 mL of 0.025M citrate-phosphate buffer containing xcex1-L-fucosidase (a product of Sigma Company, originated from bovine liver) dissolved therein, and thereafter the enzymatic reaction was conducted at 37xc2x0 C. for 60 minutes. After the enzymatic reaction, to the resulting reaction solution was added 1.0 mL of 0.2M glycine-sodium hydroxide buffer (pH 10.5), in order to terminate the enzymatic reaction. Thereafter, the absorbance (designated as xe2x80x9caxe2x80x9d) of light at 405 nm of the resulting reaction solution was measured. Concurrently, measurement was made of the absorbance (designated as xe2x80x9cbxe2x80x9d) of light at 405 nm of the reaction solution as obtained in the control test where the enzymatic reaction was conducted without addition of the test compound. Further, measurements were made of the absorbance (designated as xe2x80x9caxe2x80x2xe2x80x9d) and the absorbance (designated as xe2x80x9cbxe2x80x2xe2x80x9d) of light at 405 nm of the reaction solutions as obtained in the blank tests where the enzymatic reaction had not been conducted in the assay test, and also in the control test for the blank tests, respectively.
The rate of inhibition to xcex1-L-fucosidase was calculated by the equation:
[1xe2x88x92(axe2x88x92axe2x80x2)/(bxe2x88x92bxe2x80x2)]xc3x97100.
The concentration of the test compound capable of exhibiting 50% inhibition to the enzyme was estimated as the value of IC50. The test results obtained are summarized in Table 1 given hereinafter.
This Test Example is a test example for determining the xcex1-glucosidase or xcex2-glucosidase-inhibitory activity of the compounds according to this invention.
The evaluation of the xcex1- or xcex2-glucosidase-inhibitory activity was conducted according to a modification of the method described in the xe2x80x9cAgricultural and Biological Chemistryxe2x80x9d, 26, p.203 (1962). Namely, the test was conducted in the same manner as the test for the enzyme-inhibitory activity described in Test Example 1, except that either xcex1-glucosidase originated from yeast, or xcex2-glucosidase originated from almond (both enzymes are products of Sigma Company) was used as the glycosidase enzyme, and that p-nitrophenyl xcex1-D-glucoside or p-nitrophenyl xcex2-D-glucoside was used as the substrate. Furthermore, the enzyme-inhibitory activity was measured in the same manner as in Test Example 1, and the 50% inhibitions of the tested compounds against the enzymes were calculated, respectively. The results obtained are shown in Table 1 below.
As shown in Table 1, the compound having the formula (Ia), (Ib), (Ic), (Id) or (Ie) according to the first invention is able to inhibit strongly xcex1-L-fucosidase, xcex1-D-glucosidase and xcex2-D-glucosidase. Therefore, the compounds having the formulae (Ia), (Ib), (Ic), (Id) and (Ie) respectively according to this invention each are significantly effective as an inhibitor against said enzymes.
Moreover, it can be presumed that the compound having the formula (Ia), (Ib), (Ic), (Id) or (Ie) according to this invention has not only an activity to inhibit such glycosidases which participate in the mechanism of metastasis of cancer cells, the mechanism of inflammation and the mechanism of infection of an AIDS virus in mammalian animals, but also an activity to inhibit such glycosidases having a catabolism which participate in the digestive mechanism of food. In this view, the compound of this invention can be expected to be useful as an inhibitor to metastasis of cancer cells, which may be used for the therapeutic treatment of cancer; as an anti-inflammatory agent which may be used for treatment of chronic articular rheumatism; as an inhibitor to infection of an AIDS virus; and as an antidiabetic agent or an agent for obesity. Moreover, the compound of this invention is useful as a reagent for studying the functions of glycosidases in the living body.
Thus, according to the second aspect of this invention, there is provided a glycosidase inhibitor consisting of the compound represented by the general formula (I) or a salt thereof.
Next, there is described a process for the preparation of (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol and a (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol which are represented by the general formula (I).
In the preparation of such (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol or a (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol represented by the general formula (I) according to the first aspect of this invention, there is used as a first starting material, a (2S,3S,4R)-5-N-proteccted-2,3,4-O-tri-protected-5-aminoentane-1,2,3,4-tetraol represented by the general formula (II): 
wherein R3 is an amino-protecting group, R4 is a hydroxyl-protecting group, and X1 and X2 each are a hydroxyl-protecting group, or X1 and X2 together denote one divalent hydroxyl-protecting group (which is an alkylidene group or an aralkylidene group) represented by the formula: 
where R5 and R6 may be the same or different and each denote a hydrogen atom, an alkyl group or an aryl group, especially phenyl group, or X1 and X2 together denote a cycloalkylidene group or tetrahydropyranylidene group.
The compound of the general formula (II) may be prepared according to the method of Nishimura et al. as described in the xe2x80x9cJ. Am. Chem. Soc.xe2x80x9d, 110, p.7249-7250 (1988) and the xe2x80x9cBull. Chem. Soc. Jpn.xe2x80x9d, 65, p.978-986 (1996), as well as the method of Kudoh et al. as described in the xe2x80x9cJ. Antibioticsxe2x80x9d, 45, p.954-962 (1992).
The compound of the general formula (II) prepared according to the method as described above is employed as the starting compound and may be chemically converted in a plural of reaction steps, so that (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol and (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diols of the general formula (I) can be prepared.
Therefore, according to the third aspect of this invention, there is provided a process for the preparation of (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol or a (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol represented by the general formula (I): 
wherein R1 and R2 each are a hydrogen atom, or R1 is a hydrogen atom and R2 is a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group, or R1 and R2 together denote phthaloyl group, characterized in that the process comprises:
eliminating the hydroxyl-protecting group (R4) at the 4-position of a (2S,3S or 3R,4R)-5-N-protected-2,3,4-O-tri-protected-5-aminopentane-1,2,3,4-tetraol represented by the following general formula (II): 
wherein R3 is an amino-protecting group, R4 is a hydroxyl-protecting group, and X1 and X2 each are a hydroxyl-protecting group, or X1 and X2 together denote one divalent hydroxyl-protecting group having the formula: 
where R5 and R6 may be the same or different and each denote a hydrogen atom, an alkyl group or an aryl group, especially phenyl group, or X1 and X2 together denote a cycloalkylidene group or tetrapyranylidene group, to give a (2S,3R,4R)-5-N-protected-2,3-O-di-protected-5-aminopentane-1,2,3,4-tetraol represented by the general formula (III): 
wherein R3, X1 and X2 have the same meanings as above;
protecting the hydroxyl group at the 1-position of the compound of the general formula (III) with a hydroxyl-protecting group (R7), to prepare a (2S,3R,4R)-5-N-protected-1,2,3-O-tri-protected-5-aminopentane-1,2,3,4-tetraol represented by the general formula (IV): 
wherein R3, X1 and X2 have the same meanings as above and R7 denotes a hydroxyl-protecting group;
oxidizing the hydroxyl group at the 4-position of the compound of the general formula (IV), to prepare a (2S,3S)-5-N-protected-1,2,3-O-tri-protected-4-keto-5-aminopentane-1,2,3-triol represented by the general formula (V): 
wherein R3, X1, X2 and R7 have the same meanings as above;
subjecting the oxo group at the 4-position of the compound of the general formula (V) to the Wittig reaction, so as to convert said oxo group into a methylene group, thereby producing a (2S,3S)-5-N-protected-1,2,3-O-tri-protected-4-methylene-5-aminopentane-1,2,3-triol represented by the general formula (VI): 
wherein R3, R7, X1 and X2 have the same meanings as above;
eliminating the hydroxyl-protecting group (R7) at the 1-position of the compound of the general formula (IV), to prepare a (2S,3S)-5-N-protected-2,3-O-di-protected-4-methylene-5-aminopentane-1,2,3-triol represented by the general formula (VII): 
wherein R3, X1 and X2 have the same meanings as above;
oxidizing the hydroxyl group at the 1-position of the compound of the general formula (VII), with accompanying cyclization, to prepare a (2R,3R,4S)-1-N-protected-3,4-O-di-protected-5-methylenepiperidine-2,3,4-triol represented by the general formula (VIII): 
wherein R3, X1 and X2 have the same meanings as above;
conducting the Mitsunobu reaction on the hydroxyl group at the 2-position of the compound of the general formula (VIII) by reaction with phthalimide, to prepare a (2S,3S,4S)-1-N-protected-3,4-O-di-protected-5-methylene-2-phthalimidopiperidine-3,4-diol represented by the general formula (IX): 
wherein R3, X1 and X2 have the same meanings as above;
reducing the double bond at the 5-position of the compound of the general formula (IX), to convert the methylene group into a methyl group, thereby preparing a (2S,3S,4R,5R)-1-N-protected-3,4-O-di-protected-5-methyl-2-phthalimidopiperidine-3,4-diol represented by the general formula (X): 
wherein R3, X1 and X2 have the same meanings as above;
eliminating the phthaloyl group from the 2-amino group of the compound of the general formula (X) by treatment with hydrazine or an acid, to prepare a (2R,3S,4R,5R)-2-amino-1-N-protected-3,4-O-di-protected-5-methylpiperidine-3,4-diol represented by the general formula (XI): 
wherein R3, X1 and X2 have the same meanings as above;
protecting the amino group at the 2-position of the compound of the general formula (XI) with a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group (R2), to prepare a compound represented by the general formula (XII): 
wherein R3, X1 and X2 have the same meanings as above, R1 is a hydrogen atom and R2 is a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group; and then
eliminating both the imino-protecting group (R3) and the hydroxyl-protecting groups (X1 and X2) of the compound of the general formula (XII), or alternatively eliminating the imino-protecting group(s) and the hydroxyl-protecting groups (X1 and X2) immediately from the compound of the general formula (X) or (XI), to give the compound of the formula (I).
In carrying out the process according to the third aspect of this invention, first of all, the (2S,3S,4R)-5-N-proteccted-2,3,4-O-tri-protected-5-aminopentane-1,2,3,4-tetraol of the general formula (II) is treated with an acid or a base in an organic solvent such as tetrahydrofuran and others. Alternatively, the compound of the general formula (II) where R4 is an aralkyl group is reduced, or the compound of the general formula (II) where R4 is silylether group is treated with a fluoride such as tertabutylammonium fluoride. Thereby, the hydroxyl-protecting group (R4) at the 4-position of the compound of the general formula (II) is eliminated, to give the (2S,3R,4R)-5-N-proteccted-2,3-O-di-protected-5-aminopentane-1,2,3,4-tetraol of the general formula (III).
Then, the compound of the general formula (III) thus obtained is treated with a halogenated alkylsilane such as tert-butyl dimethylsilyl chloride and trimethylsilyl chloride, an alkoxyalkyl halide such as methoxyethoxymethyl chloride and methoxymethyl chloride, an alkyl halide, an aralkyl halide, an acid chloride such as acetyl chloride, or an acid anhydride such as acetic anhydride, in the presence of a base such as imidazole, diisopropylethylamine, triethylamine and pyridine, in a solvent such as N,N-dimethylformamide, methylene chloride, chloroform and tetrahydrofuran. Alternatively, the compound of the general formula (III) is treated with 2,3-dihydro-2H-pyran, 2,2-dimethoxypropane or others in the presence of an acid such as p-toluenesulfonic acid and others, in an organic solvent. Thereby, the hydroxyl group at the 1-position of the compound of the general formula (III) may be protected by a hydroxyl-protecting group (R7), preferably tert-butyldimethylsilyl group, to give a (2S,3R,4R)-5-N-protected-1,2,3-O-tri-protected-5-aminopentane-1,2,3,4-tetraol of the general formula (IV).
Then, the compound the general formula (IV) thus obtained is treated with an oxidizing agent such as ruthenium tetraoxide, Dess-Martin Periodinane, manganese dioxide and a confirmation of oxalyl chloride with dimethyl sulfoxide, in a solvent such as methylene chloride, chloroform, carbon tetrachloride and tetrahydrofuran. Thereby, the hydroxyl group at the 4-position of the compound of the general formula (IV) may be oxidized, to give the (2S,3S)-5-N-protected-1,2,3-O-tri-protected-4-keto-5-aminopentane-1,2,3-triol of the general formula (V).
Subsequently, the compound of the general formula (V) so obtained is treated with methylenetriphenylphosphorane in a solvent such as tetrahydrofuran, methylene chloride, benzene, acetonitrile and N,N-dimethylformamide, to convert the oxo group at the 4-position thereof into the methylene group by the resulting wittig reaction. Thereby, the (2S,3S)-5-N-protected-1,2,3-O-tri-protected-4-methylene-5-aminopentane-1,2,3-triol of the general formula (VI) is prepared.
Thereafter, the compound of the general formula (VI) thus obtained is treated with an acid or a base in an organic solvent such as tetrahydrofuran and others. Alternatively, the compound of the general formula (VI) where R4 is an aralkyl group may be reduced, or the compound of the general formula (VI) where R4 is silylether group may be treated with a fluoride such as tertabutylammonium fluoride. Thereby, the hydroxyl-protecting group (R7) at the 1-position of the compound of the general formula (VI) is eliminated, to give the (2S,3S)-5-N-proteccted-2,3-O-di-protected-4-methylenepentane-1,2,3-triol of the general formula (VII). Thereafter, the compound the general formula (VII) so obtained is treated with an oxidizing agent such as ruthenium tetraoxide, Dess-Martin Periodinane, manganese dioxide and a confirmation of oxalyl chloride with dimethyl sulfoxide, in a solvent such as methylene chloride, chloroform, carbon tetrachloride and tetrahydrofuran. Thereby, the hydroxyl group at the 1-position of the compound of the general formula (VII) may oxidized and the cyclization of the compound is accompanied, to give the (2R,3R,4S)-1-N-protected-3,4-O-di-protected-5-methylenepiperidine-2,3,4-triol of the general formula (VIII).
The 2-hydroxyl group of the compound of the general formula (VIII) thus obtained is then subjected to the Mitsunobu reaction by treatment with phthalimide in a solvent such as methylene chloride, tetrahydrofuran, chloroform, benzene, acetonitrile and N,N-dimethylformamide, to give the (2S,3S,4S)-1-N-protected-3,4-O-di-protected-5-methylene-2-phthalimidopiperidine-3,4-diol of the general formula (IX): 
wherein R3, X1 and X2 have the same meanings as above.
Then, the double bond at the 5-position of the compound of the general formula (IX) so obtained is reduced in the presence of a catalyst such as palladium, platinum oxide and Raney nickel, to convert the methylene group at the 5-position thereof into methyl group, whereby the (2S,3S,4R, 5R)-1-N-protected-3,4-O-di-protected-5-methyl-2-phthalimidopiperidine-3,4-diol of the general formula (X) is prepared.
Subsequently, the compound of the formula (X) thus obtained is treated with hydrazine or an acid such as hydrochloric acid, in a solvent such as methanol, to eliminate the phthaloyl group, whereby the (2R,3S,4R,5R)-2-amino-1-N-protected-3,4-O-di-protected-5-methylpiperidine-3,4-diols represented by the general formula (XI): 
wherein R3, X1 and X2 have the same meanings as above, can be prepared.
Thereafter, the compound of the formula (XI) as obtained is treated with an agent for introducing a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group, for example, an acid anhydride such as acetic anhydride and trifluoroacetic anhydride; an acid chloride such as acetyl chloride, trifluoroacetyl chloride and trichloroacetyl chloride; an alkyl halide such as a lower alkyl bromide, or an aryl halide such as benzyl bromide, in the presence of a base such as triethylamine, pyridine and diisopropylethylamine in a solvent such as methylene chloride, chloroform and tetrahydrofuran. By using these agents for introducing a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group, the amino group at the 2-position of the compound of the formula (XI) may be protected by the lower alkanoyl group or the lower xcfx89-trihaloalkanoyl group (R2). Thereby, prepared is the compound represented by the general formula (XII): 
wherein R3, X1 and X2 have the same meanings as above, R1 is a hydrogen atom and R2 is a lower alkanoyl group or a lower xcfx89-trihaloalkanoyl group.
Then, the compound of the general formula (XII) thus obtained is either treated with an acid such as hydrochloric acid or a base such as potassium carbonate, in a solvent such as methanol, methylene chloride, chloroform and tetrahydrofuran, or reduced in the presence of a catalyst such as palladium, platinum oxide and Raney nickel, depending on the sort of the protecting groups (R3, X1 and X2), thereby to eliminate both of the imino-protecting group (R3) and the hydroxyl-protecting groups (X1 and X2). Alternatively, immediately from the compound of the general formula (X) or (XI), both of the imino-protecting group and the hydroxyl-protecting groups (X1 and X2) may be eliminated by the same deprotection method as above. Thus, there can be produced said (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol or (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol of the general formula (I): 
wherein R1 and R2 have the same meanings as above.
When the compound of the general formula (I) so prepared by the process according to the third aspect of this invention is obtained in the form of its acid addition salt such as hydrochloride, an aqueous solution of such acid addition salt may be treated with a cation-exchange resin, for example, Dow X 50W (a product of Dow Chemical Co., U.S.A)(H+ type) according to conventional procedures, or may be purified by a chromatography using a solvent system containing ammonia, to obtain the compounds of the formula (I) in the form of the free base.
Furthermore, as will be apparent from Test Examples 1 to 2 as described hereinbefore, each of the (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol or (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol of the general formula (I), which each are a novel compound according to this invention, has the glycosidase-inhibitory activity. Therefore, all of these novel piperidine derivatives, owing to its glycosidase-inhibitory activity, is useful as an inhibitor to the metastasis of cancer cells and also is useful for the therapeutic treatment or prevention of diabetes and obesity. Furthermore, the novel piperidine derivatives according to this invention may be admixed with a conventional and pharmaceutically acceptable solid or liquid carrier to be formulated into a pharmaceutical composition.
According to the fourth aspect of this invention, therefore, there is provided a pharmaceutical composition comprising as an active ingredient (2R,3S,4R,5R)-2-amino-5-methylpiperidine-3,4-diol or a (2S,3S,4R,5R)-2-N-substituted-2-amino-5-methylpiperidine-3,4-diol of the general formula (I), or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
The pharmaceutical composition according to the fourth aspect of this invention has a glycosidase-inhibitory activity and may be administered as a medicine to animals, including human. Particularly, the pharmaceutical composition according to the fourth aspect of this invention is effective in the therapeutic treatment of cancers, or the therapeutic treatment and prevention of diabetes or obesity.
In the pharmaceutical composition according to the fourth aspect of this invention, the carrier to be mixed may be a solid or liquid carrier which is used conventionally in pharmaceutical techniques. The solid carrier may be, for example, starch, lactose, crystalline cellulose or calcium carbonate, and the liquid carrier may be, for example, physiological saline, aqueous ethanol or ethanol. The proportion of the novel piperidine derivative contained as an active ingredient in the composition is not limited, as far as it is sufficient to treat diseases, but it may be, for example, in a range of from not less than 0.01% to less than 100%, preferably in a range of from not less than 0.1% to less than 80%, based on the total weight of the composition.
When the pharmaceutical composition according to the fourth aspect of this invention is administered to the patients, it may be formulated according to conventional procedures, depending on various sorts of carriers used, the mode of administration or the medicinal form used. The formulations for oral administration include tablets, pills, granules, capsules, powders, liquids, suspensions, syrups, sublingual medicine, etc. Moreover, the formulations for parenteral administration include injections, percutaneous absorbents, inhalations, suppositories, and others. In the formulation, additives for medicines, such as surface active agents, excipients, stabilizers, wetting agents, disintegrating agents, dissolution auxiliary agents, isotonic agents, buffer agents, colorants and flavors may suitably be incorporated.
The optimal dosage of the novel piperidine derivative of this invention to be used as a medicine may vary depending on age and body weight of the patients, the type and conditions of disorders to be treated, and the route of administration employed. However, when orally administered to human, the dosage may be in a range of 1.0 to 1000 mg/kg per day for one adult. When intravenously administered, the piperidine derivative may similarly be administered in a range of 1.0 to 100 mg/kg.
Furthermore, the compound of the general formula (IX), which is prepared as an intermediate in a step of the preparation process according to the third aspect of this invention, is a novel compound and is useful for the preparation of the compound of the general formula (I) according to the first aspect of this invention. According to the fifth aspect of this invention, therefore, there is provided a (2S,3S,4S)-1-N-protected-3,4-O-di-protected-5-methylene-2-phthalimido-piperidine-3,4-diol represented by the general formula (IX): 
wherein R3 is an imino-protecting group, especially tert-butoxycarbonyl group, and X1 and X2 each are a hydroxyl-protecting group, or X1 and X2 together denote a divalent hydroxyl-protecting group, especially isopropylidene group, a cycloalkylidene group or tetrapyranylidene group.